Method for treating pulmonary fibrosis using s100a3 protein

ABSTRACT

The invention is directed to a method for diagnosing and treating a pulmonary lung disease by detecting a mutant S100A3 protein associated with pulmonary lung disease and by treating a subject with a functional S100A3 protein.

REFERENCE TO A SEQUENCE LISTING

In accordance with 37 CFR § 1.52(e)(5), the present specification makesreference to a Sequence Listing submitted electronically as a .txt filenamed “513629US_ST25.txt” on Oct. 9, 2018. The .txt file was generatedon Sep. 19, 2018 and is 9 kb in size. The entire contents of theSequence Listing are herein incorporated by reference.

BACKGROUND Field of the Invention

The invention involves the fields of molecular genetics, diagnostic andtherapeutic medicine, and pharmacology.

Description of Related Art

The “background” description provided herein is for the purpose ofgenerally presenting a context for the disclosure. Work of the presentlynamed inventor(s), to the extent it is described in this backgroundsection, as well as aspects of the description which may not otherwisequalify as prior art at the time of filing, are neither expressly orimpliedly admitted as prior art against the present invention.

The Interstitial Lung Diseases (ILDs) are a heterogeneous group ofdisorders of largely unknown etiology. They are characterized byvariable types of interstitial and alveolar inflammation, parenchymalremodeling, and fibrosis; Nogee L M. Genetics of pediatric interstitiallung disease. Current opinion in pediatrics 2006; 18:287-92. The mostcommon form of ILD is idiopathic Pulmonary Fibrosis (IPF), a progressivedisorder that usually affects individuals over 55 years of age and ismanifested histopathologically by the Usual Interstitial Pneumonitis(UIP) pattern characterized by variable degrees of inflammation,honeycomb cysts, distortion of the lung architecture, fibroblastic foci,fibrosis, and marked spatial heterogeneity; Raghu G, Collard H R, Egan JJ, et al. An official ATS/ERS/JRS/ALAT statement: idiopathic pulmonaryfibrosis: evidence-based guidelines for diagnosis and management.American journal of respiratory and critical care medicine 2011;183:788-824. Because of the lack of effective treatments and the rapidprogression to respiratory failure and death, IPF remains one of theleading indications for lung transplantations worldwide; Rahu, et al.,id.; Yusen R D, Christie J D, Edwards L B, et al. The Registry of theInternational Society for Heart and Lung Transplantation: ThirtiethAdult Lung and Heart-Lung Transplant Report—2013; focus theme: age. TheJournal of heart and lung transplantation: the official publication ofthe International Society for Heart Transplantation 2013; 32:965-78.Several environmental risk factors have been implicated in thepathogenesis of IPF including cigarette smoking and chronic aspiration(Rahu, et al., id), and a genetic predisposition has been demonstrated(Marshall R P, McAnulty R J, Laurent G J. The pathogenesis of pulmonaryfibrosis: is there a fibrosis gene? The International Journal ofBiochemistry & Cell Biology 1997; 29:107-20), however, the greatmajority of cases of IPF are sporadic.

The pathogenesis of IPF remains poorly understood and progress isimpaired by the lack of an animal model that recapitulates the salientfeatures of the human disease. Recently identified kindreds with ILDdeveloping in multiple members have provided potential clues to thepathogenesis of IPF occurring in the general population; Kropski J A,Lawson W E, Young L R, Blackwell T S. Genetic studies provide clues onthe pathogenesis of idiopathic pulmonary fibrosis. Disease models &mechanisms 2013; 6:9-17. Familial Pulmonary Fibrosis (FPF), defined asidiopathic interstitial lung disease in two or more first-degreerelatives (parent, sibling, or offspring), has been attributed tononsynonymous mutations in surfactant protein A2 (SFTPA2), surfactantprotein C (SFTPC) and ATP-binding cassette A3 (ABCA3)(Lawson W E, GrantS W, Ambrosini V, et al. Genetic mutations in surfactant protein C are arare cause of sporadic cases of IPF. Thorax 2004; 59:977-80; Wang Y,Kuan P J, Xing C, et al. Genetic defects in surfactant protein A2 areassociated with pulmonary fibrosis and lung cancer. American Journal ofHuman Genetics 2009; 84:52-9), and to a common variant in the promoterof the gene encoding mucin 5B (MUC5B) that increases MUC5B expression by37.4-fold; Seibold M A, Wise A L, Speer M C, et al. A common MUC5Bpromoter polymorphism and pulmonary fibrosis. The New England Journal ofMedicine 2011; 364:1503-12.

These mutations are proposed to converge on activation of the unfoldedprotein response; Kropski, J A, et al., 2013, id. A plurality of FPFkindreds, ˜15%, have mutations in the telomerase genes, TERT and TERC,and exhibit shortened telomeres; Tsakiri K D, Cronkhite J T, Kuan P J,et al. Adult-onset pulmonary fibrosis caused by mutations in telomerase.Proceedings of the National Academy of Sciences of the United States ofAmerica 2007; 104:7552-7; Armanios M Y, Chen J J, Cogan J D, et al.Telomerase mutations in families with idiopathic pulmonary fibrosis. TheNew England Journal of Medicine 2007; 356:1317-26. Telomere shorteningis also evident in 25% of patients with sporadic IPF who do not haveidentifiable mutations in TERT or TERC; Cronkhite J T, Xing C, Raghu G,et al. Telomere shortening in familial and sporadic pulmonary fibrosis.American Journal of Respiratory and Critical Care Medicine 2008;178:729-37.

Despite these advances, the pathogenesis of sporadic IPF remainsunclear. Accordingly it is one object of the inventors to identify anddescribe herein a new mutation segregating in three siblings withpulmonary fibrosis in the calcium binding protein gene, S100A3 (NM002960). The mutation leads to lower expression of the S100A3 proteinand is associated with aberrant intracellular calcium homeostasis andreduced capacity to tolerate oxidative stress in isolated cells. Theseresults indicate that S100A3 regulates pulmonary fibrosis andrestoration of S100A3 protein levels that may reverse the clinicalsymptoms and provide a new therapy for the disease. It is a furtherobject of the present disclosure to provide a method for determiningmitigating and/or reducing a risk of fibrosis.

BRIEF SUMMARY OF THE INVENTION

The invention involves treatment of lung fibrosis and respiratoryfailure using S100A3 protein in subjects that lack functional S100A3protein, such as a homozygous subject carrying missense variantrs138355706, (229C>T). As shown herein the inventors identified ahomozygous variant in a previously unreported gene coding for thecalcium binding protein S100A3, segregating in the 3 patients afflictedand 13 additional family members who were either heterozygous carriersor wild-type normal for the variant. Indirect immunofluorescence andWestern blots demonstrated decreased expression of the protein in thelungs of patients and in cells isolated from the patient's skin. Thiswas concomitant with aberrant calcium homeostasis in isolated patient'sfibroblasts. The introduction of wild type gene in the patient's cellsrestored their normal calcium responses. Furthermore, the introductionof the mutant transcript caused aberrant calcium homeostasis that wassimilar to that of seen in patient's cells.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the disclosure and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1A. The pedigree of Family 1A with haplotype and genotype analyses;disease haplotype is highlighted in yellow (or *). Genotype of variantsin genes S100A3: c.229 C>T transition (r5138355706) causing p.R77C isdenoted in red text (or see lines designated by arrows).

FIG. 1B shows linkage analysis resulting in a peak where the maximummultipoint LOD score was 5.28 corresponding to chromosome 1p12-q21.3.

FIG. 1C shows a single ROH as a result of homozygosity mapping shared byall affected patients between rs10802117 and rs11808053 confirminglinkage analysis.

FIG. 1D. Shows the sequence chromatogram indicating the wild-type,homozygous affected and heterozygous carrier forms of the C to Ttransition at position c.229 changing arginine residue to cysteine atposition 77 of the S100A3 protein (c.229C>T, p.R77C). Mutation name isbased on the full-length S100A3 transcripts.

FIGS. 2A and 2B. Immunofluorescence micrographs demonstrating reducedexpression of S100A3 protein levels in normal (FIG. 2A) and patientslung tissues (FIG. 2B).

FIG. 2C. Expression of the S100A3 mRNA in control (first bar) andpatients cells (second bar) as measured by qtPCR analysis.

FIG. 2D. Expression of the S100A3 mRNA in control and patients asmeasured by PCR analysis illustrating the absence of wild type gene inthe patient sample and presence of only mutated form of the genes. Betaactin is used as control.

FIG. 2E. Expression of the S100A3 protein in control and patient cellsisolated from skin (fibroblasts) using western blot analysis. Beta actinis used as control.

FIG. 2F. Expression of the S100A3 transcripts in human embryonic kidneycells (HEK). Western blot analysis of cells transfected with the wildtype S100A3 (HEK-W) and mutant S100A3 (HEK-M). Beta actin is used ascontrol.

FIG. 3A. Calcium response of skin fibroblasts (isolated from control;upper green line; and patients; lower red line) to stimulation byfibroblast growth factor (FGF) added at the first arrow. Second arrowdepicts the time of addition of the calcium ionophore ionomycin and thethird arrow indicates the time of addition of EGTA.

FIG. 3B. Calcium response of skin fibroblasts (isolated from control;upper blue line; and patients, lower red line) to stimulation bybradykinin.

FIG. 3C. Maximum calcium responses to ionomycin in control, patients,patient cells transfected with wildtype transcript of S100A3 (rescued)and control cells transfected with mutant S100A3 transcript (mutant).

FIGS. 3D and 3E. 3D intensity maps of Mito Tracker® Red CMXRos labelledcells isolated from control (FIG. 3D) and patients (FIG. 3E). Maps arecolor coded so that warm colors indicate high intensity and cold colorsindicate low intensity.

FIG. 3F. Mean fluorescence intensity obtained from flow cytometry ofskin fibroblasts isolated from patient and control cells stained withMitotracker Green.

FIG. 3G. Effect of externally added oxidation (H₂O₂, 0.03%, arrow) onpatients and control cells labeled with Mito Tracker® Red CMXRos.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present disclosure will now be described more fullyhereinafter with reference to the accompanying drawings, in which some,but not all embodiments of the disclosure are shown.

The term S100A3 polypeptide describes a class of polypeptides thattypically have about 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, <100 or100% sequence identity or similarity to SEQ ID NO: 2. This classincludes a wild-type, functional S100A3 polypeptide of SEQ ID NO: 2 andother functional natural variants as well as a non-functional mutantS100A3 polypeptide of SEQ ID NO: 4. A mutant S100A3 protein will have anon-wild-type amino acid sequence, and typically exhibit aberrantexpression, folding and/or activity compared to wild-type S100A3protein. One example of such a mutant S100A3 protein is described by SEQID NO: 4 which contains the p.R77C point mutation.

BLASTN may be used to identify a polynucleotide sequence having at least70, 75, 80, 85, 90, 95, 96, 97, 98, 99, <100, or and 100% (or anyintermediate %) sequence identity to a reference polynucleotide. Arepresentative BLASTN setting optimized to find highly similar sequencesuses an Expect Threshold of 10 and a Wordsize of 28, max matches inquery range of 0, match/mismatch scores of 1/-2, and linear gap cost.Low complexity regions may be filtered/masked. Default settings aredescribed by and incorporated by reference to hypertext transferprotocol://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&BLAST_PROGRAMS=megaBlast&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome (last accessedSep. 10, 2018).

BLASTP can be used to identify an amino acid sequence having at least70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 96, 97.5%, 98%, 99%, <100%or 100% (or any intermediate %) sequence identity or similarity to areference amino acid using a similarity matrix such as BLOSUM45,BLOSUM62 or BLOSUM80 where BLOSUM45 can be used for closely relatedsequences, BLOSUM62 for midrange sequences, and BLOSUM80 for moredistantly related sequences. Unless otherwise indicated a similarityscore will be based on use of BLOSUM62. When BLASTP is used, the percentsimilarity is based on the BLASTP positives score and the percentsequence identity is based on the BLASTP identities score. BLASTP“Identities” shows the number and fraction of total residues in the highscoring sequence pairs which are identical; and BLASTP “Positives” showsthe number and fraction of residues for which the alignment scores havepositive values and which are similar to each other. Amino acidsequences having these degrees of identity or similarity or anyintermediate degree of identity or similarity to the amino acidsequences disclosed herein are contemplated and encompassed by thisdisclosure. A representative BLASTP setting that uses an ExpectThreshold of 10, a Word Size of 3, BLOSUM 62 as a matrix, and GapPenalty of 11 (Existence) and 1 (Extension) and a conditionalcompositional score matrix adjustment. Default settings for BLASTP aredescribed by and incorporated by reference to hypertext transferprotocol://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome(last accessed Sep. 10, 2018).

The term “S100A3 polypeptide” also includes functional fragments ofS100A3 which exert at least one biological, physiological orimmunological activity or function of a wild-type S100A3 polypeptide. Afragment may contain up to 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90,95, 96, 97, 98, 99 or <100% of the residues of a native or variantS100A3 polypeptide.

A S100A3 polypeptide, variant or fragment thereof may be produced bymethods known in the art including by cloning and recombinant expressionin a host cell or by chemical synthesis. Many protein expression andpurification systems are known and incorporated by reference tohypertext transfer protocolsecure://_en.wikipedia.org/wiki/Proteinproduction (last accessed Sep.10, 2018).

A S100A3 polynucleotide is one that encodes S100A3 protein or afunctional fragment thereof as well as naturally occurring polymorphs ormutants thereof. Variant S1001A3 polynucleotides have sequences that areat least 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99or <100% identical to those of SEQ ID NOS: 1 or 3. Examples include thewild-type polynucleotide described by SEQ ID NO: 1 and a mutant S100A3polynucleotide of SEQ ID NO: 3 identified by the inventors. A polymorphor variant may have 1, 2, 3, 4, 5, or more nucleotide deletions,substitutions or insertions compared to a naturally occurring S100A3polynucleotide.

Single nucleotide polymorphisms and other polymorphisms. A polymorphismin an S100A3 polypeptide may be detected or analyzed by methods known inthe art, including by methods using the polymerase chain reaction, DNAsequencing, capillary electrophoresis, mass spectrometry; single-strandconformation polymorphism (SSCP); single-base extension; electrochemicalanalysis; denaturating HPLC and gel electrophoresis; restrictionfragment length polymorphism; or hybridization analysis. Typically, thepolymorphism will occur in a coding region for the S100A3 protein, suchas a missense mutation causing an amino acid residue substitution, but apolymorphism may also occur outside of the coding region, for example,in a flanking DNA sequence.

Homozygous affected subjects typically have two copies of anon-functional mutant S100A3 gene.

Heterozygous carriers. Some individuals have one wild-type S100A3 geneand one mutant S100A3 gene and thus are heterozygous for this gene.Carrying a single copy of the nonfunctional mutant gene may causealtered responses to certain physiological stimuli and negatively impactlung function, especially lower lung functions including compromisinglung reserve.

Treatments include administering functional S100A3 protein to a subjectdeficient in or totally lacking functional S100A3 protein. As shownherein the effects of a mutation in S100A3 on cellular responses can bereversed by administration of functional, wild-type S100A3 protein.

Functional S100A3 protein may be administered systemically and/orlocally, preferably directly or indirectly into the respiratory system.Functional S100A3 protein may be administered into the lungs using anebulizer or metered dose inhaler to rectify abnormal cellular responsesor halt the progression of lung fibrosis. Functional S100A3 protein andother active ingredients, such as antibodies that selectively bind tonon-functional S100A3 protein, are preferably prepared in a form thatcan reach and persist in target respiratory tissues such as the lungs,bronchi and other airways.

A pulmonary route of administration offers many advantages includingnoninvasive delivery of protein and peptide-based drugs, absorption of adrug through the lungs by simple diffusion and carrier-mediatedtransport, often a decrease in the amount of drug to be administered,fast adsorption and better patient compliance that other parenteralroutes.

However, functional S100A3 protein, or other active ingredients likeantibodies that bind to and remove non-functional, mutant S100A3protein, may be administered by other routes including intravenously,intraperitoneally, subcutaneously, intramuscularly, and topically.

Oral administration may be contemplated so long as the functional S100A3protein is administered in a form that is not digested prior to reachinga target site or being adsorbed into the body.

A nebulizer is a drug delivery device that administered a drug, such asfunctional S100A3 or antibodies that bind to non-functional or othermedicaments in an inhalable form. Nebulizers for treatment of cysticfibrosis, asthma, COPD and other respiratory diseases are known andincorporated by reference to hypertext transfer protocolsecure://en.wikipedia.org/wiki/Nebulizer. These include soft mistinhalers, jet nebulizers, ultrasonic wave nebulizers, and nebulizersusing vibrating mesh technology.

A metered-dosage inhaler is another drug delivery device that delivers aselected or metered amount of a medication, such as functional S100A3protein or an antibody that binds to non-functional or mutant S100A3protein. Typically, this device produces and releases an aerosol ofmicrometer-sized particles that are inhaled. In some cases, theparticles may be a dry powder in others as a mist or in a semiliquidform. Metered-dose inhalers and their various components, propellants,excipients and other elements are described by and incorporated byreference to hypertext transfer protocolsecure://en.wikipedia.org/wiki/Metered-dose inhaler. An inhalablecomposition may be formulated in the form of a hydrofluoroalkane inhaleror HFA (metered dose inhaler or MDI), dry powder inhaler (DPI), or as anebulizer solution.

Functional S100A3 protein or antibodies that bind to non-functionalS100A3 protein or other medicaments may be administered as a bolus, orover a period of time, such as at 0.25, 0.5, 1, 2, 3, 4, 5 or more hourintervals. Dosages of functional S100A3 protein may range from 1, 2, 5,10, 20, 50, 100, 200, 500 or more picomoles to 1, 2, 5, 10, 20, 50, 100,200, 500 or more millimoles.

Modes of administration for functional S100A3 or other activeingredients of the invention include by nebulizer, oral, sublingual,rectal, topical, parenteral (other than IV and IM), intravenousinjection, intramuscular injection and intraperitoneally. Injection ofDNA encoding a functional S100A3 protein may also be used as well asgene therapy of cells, such as by transformation of pulmonary cells witha nucleic acid encoding functional S100A3 protein.

Intravenous administration delivers a drug, such as functional S100A3protein or an antibody that binds to non-functional S100A3 proteindirectly into a vein. It is usually considered one of the fastest waysto deliver a drug. A drug may be administered as a bolus, for example,by i.v. push or by injection with a syringe, or by continuous infusionover a period of 0.0.25, 0.5, 1, 2, 3, 4, 5 or more hours.

Antibody-based treatments include administration of antibodies thatselectively bind to non-functional S100A3 protein and inactivate,promote its removal, or remove it so that subsequently administeredwild-type or functional S100A3 protein may exert a therapeutic functionby replacing non-functional S100A3 protein. Antibodies that selectivelybind to mutant S100A3 protein, but have a lower or no binding affinityfor wild-type or functional S100A3 protein may be administeredsystemically or locally, including into the respiratory system. Thesemay be administered before, at the same time as, or after administrationof a functional S100A3 protein. In some embodiments, an antibody thatbinds to both functional S100A3 protein as well as to a non-functionalmutant S100A3 protein may be pre-administered and after a timesufficient for removal or reduction of non-functional S100A3 proteinlevels by followed by administration of a functional S100A3 protein.

An antibody may be polyclonal, monospecific, or monoclonal, preferablymonoclonal for purposes of uniformity and standardization. Antibodiesinclude IgA, IgE, IgG and IgM as well as their various subclasses.Secretory antibodies, such as sIgA or pentameric IgM may be used.Antibody fragments or antibody conjugates that retain an ability torecognize and bind to non-functional S100A3 protein may also be used.

Compositions. A functional S100A3 protein or other active ingredient ofthe invention may be formulated as a pharmaceutically acceptablecomposition, for example, with a physiologically acceptable carrier forpulmonary or intravenous administration. As used herein, a “composition”refers to a mixture of the active ingredient with at least one otherchemical component, such as a pharmaceutically acceptable carrier orexcipient. One purpose of a composition is to facilitate administrationof a functional S100A3 protein or other active ingredients of theinvention to a subject.

Depending on the intended mode of administration, the composition can bein the form of solid, semi-solid, liquid, or aerosol dosage forms, suchas solutions, powders, tablets, suppositories, pills, capsules, powders,liquids, or suspensions, preferably in unit dosage form suitable forsingle administration of a precise dosage.

The phrase “pharmaceutically acceptable” as used herein refers tocompounds, counterions, materials, compositions, and/or dosage formswhich are, within the scope of sound medical judgment, suitable for usein contact with the tissues of human beings without excessive toxicity,irritation, allergic response, or other problem or complication andcommensurate with a reasonable benefit/risk ratio. A composition istypically a combination of an active ingredient with a carrier orexcipient, inert or active, making the composition especially suitablefor diagnostic or therapeutic use in vivo, ex vivo, or in vitro.

The term active ingredient, as used herein, refers to an ingredient inthe composition that is biologically active, for example a functionalS100A3 protein, antibody that binds to non-functional or mutant S100A3protein or polynucleotide encoding these proteins. Other activeingredients include, but are not limited to, those that exert asubstantial pharmacokinetic or pharmacodynamic activity when inadmixture with the active ingredients of the invention, for example,other excipients, buffers, or stabilizers, or other drugs approved foradministration to the respiratory system.

As used herein, a pharmaceutically acceptable carrier refers to acarrier or diluent that does not cause significant irritation to anorganism, does not abrogate the biological activity and properties ofthe administered active ingredient, and/or does not interact in adeleterious manner with the other components of the composition in whichit is contained.

The term carrier encompasses any excipient, binder, diluent, filler,salt, buffer, solubilizer, lipid, stabilizer, or other material wellknown in the art for use in pharmaceutical formulations, for example,for intravenous administration a carrier may be sodium chloride 0.9% ormixtures of normal saline with glucose or mannose. The choice of acarrier for use in a composition will depend upon the intended route ofadministration for the composition. The preparation of pharmaceuticallyacceptable carriers and formulations containing these materials isdescribed in, e.g., Remington's Pharmaceutical Sciences, 21st Edition,ed. University of the Sciences in Philadelphia, Lippincott, Williams &Wilkins, Philadelphia Pa., 2005, which is incorporated herein byreference in its entirety. Examples of physiologically acceptablecarriers include buffers such as phosphate buffers, citrate buffer, andbuffers with other organic acids; antioxidants including ascorbic acidor tocopherol; low molecular weight polypeptides having 2, 3, 4, 5, 6,7, 8, 9, 10 or fewer residues; proteins, such as serum albumin, gelatin,or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as TWEEN® (ICI, Inc.;Bridgewater, N.J.), polyethylene glycol (PEG), and PLURONICS® (BASF;Florham Park, N.J.).

In some embodiments an active ingredient of the invention, such asfunctional S100A3 protein may be administered in a carrier used toadminister other drugs to the respiratory system, such as carriers foranti-inflammatory drugs. It may also be administered in combination withan anti-inflammatory drug such as an inhaled steroid such asBeclomethasone dipropionate(Qvar), Budesonide (Pulmicort),Budesonide/Formoterol(Symbicort)—a combination drug that includes asteroid and a long-acting bronchodilator drug, Fluticasone (Flovent),Fluticasone inh powder (Arnuity Ellipta), Fluticasone/Salmeterol(Advair)—a combination drug that includes a steroid and along-actingbronchodilator drug, Mometasone (Asmanex), orMometasone/formoterol (Dulera)—a combination drug that also includes along-acting bronchodilator drug. In some embodiments one or more NSAIDsmay be adminstered as additional active ingredients, these includeaspirin, celecoxib (Celebrex), diclofenac (Cambia, Cataflam,Voltaren-XR, Zipsor, Zorvolex), diflunisal (Dolobid), etodolac,ibuprofen (Motrin, Advil), indomethacin (Indocin), ketoprofen, ketorolac(Toradol), nabumetone, naproxen (Aleve, Anaprox, Naprelan, Naprosyn,oxaprozin (Daypro), piroxicam (Feldene), salsalate (Disalsate),sulindac) or tolmetin. A secondary active ingredient may be administeredby the same or by a different route or mode of administration.

For therapeutic purposes, formulations for parenteral administration canbe in the form of aqueous or non-aqueous isotonic sterile injectionsolutions or suspensions. The term parenteral, as used herein, includesintravenous, intravesical, intraperitoneal, subcutaneous, intramuscular,intralesional, intracranial, intrapulmonal, intracardial, intrasternal,and sublingual injections, or infusion techniques. These solutions andsuspensions can be prepared from sterile powders or granules having oneor more of the carriers or diluents mentioned for use in theformulations for oral administration, preferably in adigestion-resistant form such as an enteric coating. The activeingredient can be dissolved in water, polyethylene glycol, propyleneglycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil,benzyl alcohol, sodium chloride, and/or various buffers. Other adjuvantsand modes of administration are well and widely known in thepharmaceutical art.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions can be formulated according to the known artusing suitable dispersing or wetting ingredients and suspendingingredients. The sterile injectable preparation can also be a sterileinjectable solution or suspension in a non-toxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that can be employed are water,Ringer's solution, and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil can be employedincluding synthetic mono- or diglycerides. In addition, fatty acids,such as oleic acid, find use in the preparation of injectables. Dimethylacetamide, surfactants including ionic and non-ionic detergents,polyethylene glycols can be used. Mixtures of solvents and wettingingredients such as those discussed above are also useful.

DNA- or RNA-based vaccines. In some embodiments, DNA encoding an S100A3protein of the invention may be replicated or synthesized by knownmethods. The DNA is then formulated for administration to a subject, forexample, by pulmonary, intravenous, subcutaneous, intramuscular,intrapulmonary, or intralymphatic administration. DNA-based vaccines andmethods of their use are known and are incorporated by reference toTregoning, J S, et al., Using Plasmids as DNA Vaccines for InfectiousDiseases. Microbiol Spectr. 2014 December; 2(6). doi:10.1128/microbiolspec.PLAS-0028-2014; Ramirez, L A, et al., Therapeuticand prophylactic DNA vaccines for HIV-1. Expert Opin Biol Ther. 2013April; 13(4):563-73. doi: 10.1517/14712598.2013.758709; Williams, J A,Improving DNA vaccine performance through vector design. Curr Gene Ther.2014; 14(3):170-89.

In some embodiments, mRNA encoding a S100A3 polypeptide of the inventionmay be produced by transcribing or otherwise producing an RNA moleculecorresponding to DNA encoding the polypeptide by known methods. The RNAis then formulated for administration to a subject, for example, byintravenous, subcutaneous, intramuscular, intrapulmonary, orintralymphatic administration. RNA-based vaccines and methods of usingthem to induce immunity are described by and incorporated by referenceto Hubaud, A., RNA vaccines: a novel technology to prevent and treatdisease, hypertext transferprotocol://_sitn.hms.harvard.edu/flash/2015/rna-vaccines-a-novel-technology-to-prevent-and-treat-disease/and to Pardi, N., et al., mRNA vaccines—a new era in vaccinology Nat RevDrug Discov. 2018 April; 17(4):261-279. doi: 10.1038/nrd.2017.243. Epub2018 Jan. 12.

Other Active Ingredients.

In some embodiments, other active ingredients in addition to afunctional S100A3 protein or polynucleotide encoding it, or to anantibody or antibody fragment that binds to non-functional S100A3protein may be incorporated into a composition or separatelyadministered in conjunction with at least one active ingredient of theinvention. These include anti-inflammatory agents, colchicine,corticosteroids, including inhalable corticosteroids likeflunisolide⋅fluticasone furoate⋅fluticasone propionate⋅triamcinoloneacetonide⋅beclomethasone dipropionate and Budesonide; immunosuppressantssuch as cyclophosphamide, azathioprine, methotrexate, penicillamine, andcyclosporine, or cytokines, such as IFN-gamma.

Protease inhibitors may be administered to increase the biological lifeof functional S100A3 protein or other active ingredients of theinvention or otherwise inhibit protease activity during treatment; seehypertext transfer protocolsecure://www.ddw-online.com/therapeutics/p148402-protease-inhibitor-therapeutics-for-respiratory-disease-winter-03.html,last accessed Sep. 10, 2018, incorporated by reference).

In some embodiments, one or more chaperonins may be administered tocorrect protein misfolding, along with an active component of theinvention such as along with a functional S100A3 protein. Chaperoninsare incorporated by reference to Elena L. Rudashevskaya, ThomasStockner, Michael Trauner, Michael Freissmuth and Peter Chiba.Pharmacological correction of misfolding of ABC proteins. Drug DiscovToday Technol. 2014 June; 12(100): e87-e94. doi:10.1016/j.ddtec.2014.03.009. PMCID: PMC4039138 PMID: 25027379. Inanother embodiment, the mutant S100A3 proteins described herein may beused as targets for screening of chaperonin compounds that increase orrestore wild-type S100A3 functionality. Such a method would involvecontacting a mutant S100A3 protein or a cell expressing it with achaperonin, detecting an increase in at least one wild-type S100A3activity, and, optionally, treating a subject who expresses a mutantS100A3 protein with a chaperonin that increases the wild-type activity.

Antioxidants, such as ascorbates or tocopherols may be administered aswell as oxygen before, during or after therapy with an active ingredientof the invention.

Improvement in status of pulmonary fibrosis may be determined by CTscanning to show general fibrosis and staging according to CT resultsand lung function tests. Improvement is measured by halting the progressof the disease or by reducing the already existing fibrosis which can bedetermined using CT scans and lung functional tests.

Other, non-limiting embodiments of the invention include those describedbelow.

A method for determining a risk of fibrosis comprising detecting amutant S100A3 protein or a mutant polynucleotide encoding it in abiological sample from a subject, and, optionally, treating the subjectto reduce a risk of fibrosis or treating the subject for fibrosis. Thismethod may include detecting the mutant S100A3 protein and wherein themutant S100A3 protein comprises an amino acid residue that is notarginine at position 77; include detecting the mutant S100A3 protein andwherein the mutant S100A3 protein comprises an amino acid residue thatis cysteine at position 77; detecting a mutant S100A3 protein and themutant S100A3 protein comprises the amino acid sequence described by SEQID NO: 4; or detecting a mutant S100A3 protein by contacting it with anantibody that selectively binds to the mutant S100A3 protein incomparison to a not mutated S100A3 protein.

An antibody to a mutant S100A3 protein, such as one that binds to S100A3(SEQ ID NO: 4) carrying the p.R77C mutation, will preferably have ahigher affinity or avidity for the mutant S100A3 protein than for thewild-type protein, more preferably, will bind to the mutant S100A3protein but will substantially not bind to the wild-type S100A3 protein.Linear B cell epitopes of a mutant S100A3 protein will typically containone or more mutations to the wild-type amino acid sequence, for example,the p.R77C mutation shown by SEQ ID NO: 4. In some instances, a mutationwill be part of, of form by its allosteric effects, a conformationalepitope that can be used to distinguish a wild-type S100A3 protein froma mutant S100A3 protein. B cell epitopes typically contain about 5 to 20amino acid residues Those skilled in the art can identify B cellepitopes by methods known in the art including those described byEl-Manzalawy Y, et al., Comput Syst Bioinformatics Conf. 2008; 7:121-132. A B cell epitope on a mutant S100A3 protein may span 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid residuesof SEQ ID NO: 4 and contain the p.R77C mutation or another mutation toresidue 77 (or other residues) of wild-type S100A3 protein. Peptideshaving B cell epitopes may be used to produce polyclonal or monoclonalantibodies to a mutant S100A3 protein by methods known in the artincluding those described by or cited to by hypertext transfer protocolsecure://en.wikipedia.org/wiki/Monoclonal_antibody (incorporated byreference, last accessed Sep. 23, 2018).

In other embodiment this method may include detecting a polynucleotideencoding a mutant S100A3 protein and wherein the mutant S100A3 proteincomprises an amino acid residue that is not arginine at position 77;detecting a polynucleotide encoding a mutant S100A3 protein and whereinthe mutant S100A3 protein comprises an amino acid residue that iscysteine at position 77; detecting a polynucleotide encoding a mutantS100A3 protein that comprises the amino acid sequence described by SEQID NO: 4; detecting a polynucleotide comprising rs138355706 (229C>T); ordetecting a polynucleotide that encodes the mutant S100A3 protein usingprobes and/or primers that selectively recognize the polynucleotidecomprising rs138355706 (229C>T). Primers that amplify a polynucleotideencoding a mutant S100A3 protein may be designed by methods known in theart, such as those disclosed by Primer-BLAST available at hypertexttransfer protocol secure://www.ncbi.nlm.nih.gov/tools/primer-blast(incorporated by reference, last accessed Sep. 23, 2018). Primers andprobes that identify polynucleotides encoding mutant S100A3 proteins mayalso be designed using tools such as OligoArchitect™ tool available fromSigma-Aldrich and incorporated by reference to hypertext transferprotocolsecure://www.sigmaaldrich.com/technical-documents/articles/biology/oligoarchitect-online.html (last accessed Sep. 23, 2018). Those skilled in the art can designprimers and probes for amplification and/or detection of polynucleotidesencoding S100A3 mutant polypeptides using these tools and then selectprimers that preferentially amplify or hybridize to polynucleotidesencoding mutant S100A3 protein in comparison to those encoding thewild-type S100A3 protein. One embodiment of primers or probes of theinvention encompasses any primer or probe that can detect the c.229C>Tsubstitution as shown in SEQ ID NO: 3 in any order.

The method may further include detecting whether the subject ishomozygous for a polynucleotide encoding a mutant S100A3 protein,heterozygous for a polynucleotide encoding a mutant S100A3 protein, orwhether the subject is homozygous for a gene encoding a not mutatedS100A3 protein. In some embodiments, the method may involve determininga pedigree or degree of relationship for a heterozygous or homozygoussubject.

The method may involve treating a fibrosis, including organ fibrosis orpulmonary fibrosis.

Another embodiment of the invention is directed to a method for reducingthe risk of fibrosis or lung disease, or for treating fibrosis or lungdisease in a subject who has at least one gene encoding a non-functionalmutant S100A3 protein comprising administering to said subject apolynucleotide encoding a functional S100A3 protein, administering afunctional S100A3 protein, or administering at least one activator orinhibitor of an S100A3 protein. The subject in this method may be onewho has at least one gene comprising a missense mutation rs138355706,(229C>T). In some embodiments, the administering in this method includesadministering a polynucleotide encoding a functional S100A3 protein intothe respiratory system; administering a functional S100A3 protein intothe subject's lungs and further comprises administering an antibody orantibody fragment that specifically binds to the mutant S100A3polypeptide of SEQ ID NO: 4; or administering at least one activator orinhibitor of S100A3 protein into the subject's lungs.

Another embodiment of the invention is directed to a pharmaceuticalcomposition containing at least one polynucleotide encoding a functionalS100A3 protein, at least one functional S100A3 protein, or at least oneactivator or inhibitor of an S100A3 protein, in an amount sufficient toreduce the severity of fibrosis when administered to a subject in needthereof. Such a composition may be in the form of a dust, aerosol ormist or another form that reaches the lungs or other target site onceinhaled.

Example

The following Example illustrates various aspects of the presentinvention. They are not to be construed to limit the claims in anymanner whatsoever.

Brief Case Description.

Three siblings, two girls and one boy, from a total of seven childrenwere born healthy to consanguineous Saudi Arabian parents after normalpregnancies and deliveries (see chart in FIG. 1A). The parents did notreport any medical problems until all three children developedrespiratory fibrosis at a young age. All 3 affected siblings had anidentical clinical presentation and course. They all developed dyspneain their early teens and apart from the lung abnormalities, complete andextensive medical examinations revealed normal appearance, developmentand laboratory findings.

Pulmonary function testing showed severe restriction and impaired oxygentransfer (FIG. 2B) and fibrosis with a non-specific pattern was revealedby chest CT imaging (FIG. 2A). The subjects had extensive medicalinvestigations in Saudi Arabia and the USA, resulting in exclusion ofknown (including genetic) causes of lung fibrosis. Their respiratorysymptoms worsened with age and all 3 patients developed chronic type 2respiratory failure.

Two of the siblings underwent lung transplantation at ages 22 years and23 years of age, but died due to primary graft failure. The thirdsibling died from respiratory failure at 23 years of age. A lung autopsyfrom one of the subjects revealed nonspecific fibrosis like pattern withmicroscopic honeycombing (FIG. 1C).

Methods.

All subjects provided and institutional review board approved writteninformed consent (KFSH&RC-RAC #2120 009).

Sequence Analysis.

Genomic DNA was extracted from whole blood or paraffin-embedded archivedtissue of affected patients, their parents and 11 unaffected relativesusing standard methods. Bi-directional exomic sequencing of the codingregions of known IPF-associated genes (TERT, TERC, ABCA3 and SFTPB) wasperformed in patients and nuclear family members. Sequence analysis wasperformed manually using the SeqMan 6.1 module of the Lasergene (DNAStar Inc. Wis., USA) software package.

Linkage analysis and homozygosity mapping. Linkage analysis wasperformed using the Allegro module of the easyLINKAGE; Lindner T H,Hoffmann K. easyLINKAGE: a PERL script for easy and automatedtwo-/multi-point linkage analyses. Bioinformatics 2005; 21:405-7(incorporated by reference). This was followed by genotyping on allmembers using the Affymetrix Axiom® Genome-Wide CEU 1 Array platformanalyzed by homozygosity mapping using AutoSNPa. Direct sequencing ofcandidate genes in the ROH, linkage interval and exome re-sequencingdata of genomic DNA was performed using primer pairs designed togenerate overlapping PCR amplicons of the entire coding region of eachgene.

Detection of S100A3 Message and Protein Levels.

The expression of S100A3 mRNA was performed using RNA isolation kits;TRIzol reagent (Ambion, Grand Island, N.Y.), and complementary DNA wassynthesized from 1-5 μg of RNA cDNA kit (Qiagen, Germantown, Md.)according to the manufacturer's protocol. Primers for 18S rRNA werepurchased from SABiosciences/Qiagen (Valencia, Calif.). Primers forS100A3 (forward: 5′-cccgaactggtcaactctca (SEQ ID NO: 5); reverse:5′-gcctggcagagcttgtattt (SEQ ID NO: 6)), and plasmid backbone (forward:5′-gtggcgctttctcatagctc (SEQ ID NO: 7); reverse: 5′-tgtcttaccgggttggactc(SEQID NO: 8) were designed using PRIMER3. SABiosciences/Qiagen(Valencia, Calif.). qRT-PCR was performed on an Applied BiosystemsStepOne Plus PCR system (Carlsbad, Calif.) using SYBR Green qRT-PCRMastermix (SABiosciences) according to the manufacturer's directions.For qRT-PCR of S100A3 in control and patient samples the followingprimers were used: (forward: 5′-ggaccccgactgagtttcg (SEQ ID NO: 9);reverse: 5′-gctctgaggggcagtccttg (SEQ ID NO: 10). For GAPDH (control forqRT-PCR) the following primers were used (forward:5′-caccatcttccaggagtgag (SEQ ID NO: 11); reverse:5′-tcacgccacagtttcccgga (SEQ ID NO: 12)).

Immunofluorescence and western blots were performed using using primaryrabbit antibodies against S100A3 purchased from Santa Cruze (SantaCruze, Calif.), followed by peroxidase-conjugated goat anti-rabbit IgG(Jackson ImmunoResearch Laboratories; West Grove, Pa.).

Intracellular Calcium and Mitochondrial Integrity Measurements.

Intracellular calcium measurements were performed on patientfibroblasts, control fibroblasts (from unaffected donors) transfectedwith mutant-transcript of S100A3, patients fibroblasts transfected withwild-type S100A3 and control fibroblasts from unaffected individuals asdescribed previously. Receptor mediated changes in intracellularfluorescence intensity in response to FGF (10 ng/ml) and ionomycin (2μM) were followed using Zeiss LSM 510 META laser scanning confocalsystem (Carl Zeiss MicroImaging, GmbH, Germany). Mitochondria stainingwere performed using Mito Tracker® Red CMXRos (2 μM, Invitrogen™Molecular Probes™, USA) and viewed under Zeiss Yokogawa Spinning Diskconfocal system (Carl Zeiss MicroImaging, GmbH, Germany).

Identification of a Novel IPF Region on Chromosome 1.

Bi-directional exomic sequencing of the candidate familialILD-associated genes, TERT, TERC, ABCA3 and SFTPB, did not reveal anyvariations compared to the normal reference sequences in the patients orfamily members. Subsequent sequence and linkage analysis identified asingle peak with a multipoint LOD score of 2.95 corresponding tochromosome 1p12-q23.1 (FIG. 1B). This was confirmed by homozygositymapping (Carr I M, Flintoff K J, Taylor G R, Markham A F, Bonthron D T.Interactive visual analysis of SNP data for rapid autozygosity mappingin consanguineous families. Human Mutation 2006; 27:1041-6, incorporatedby reference), which corroborated a single ROH (FIG. 1C) that was sharedby all 3 affected patients and not any unaffected family members andincluded 1579 SNP calls with identical genotypes between rs10802117 andrs1615480 (chrl:120,127,864-158,944,584 bp; base numbering is accordingto UCSC Genome Browser, build hg19) spanning approximately 38 Mbcontaining over 800 annotated genes. Three genes within this region,(MUC1, SMG5, and BCAN) were identified, sequenced and excluded aspotential candidate familial ILD-causing genes based on their similarityin function, expression, and/or protein family, type to previouslyidentified familial ILD-associated genes.

Exome Sequencing.

Whole exome sequencing of all genes in the 31.3 Mbp linkage region wasperformed in the proband (Individual IV:1 FIG. 1A). After filtering forhomozygous non-synonymous SNVs that were either novel or had either lowor unknown minor allele frequency in dbSNP, only 3 previously describedvariants were identified, rs3795737 in ISG20L2, rs143224912 in SETDB1,and rs138355706 in S100A3. Rs3795737 was excluded because it was foundin the homozygous state throughout many different populations. Exomicsequencing of SETDB1 and S100A3 was performed in all 3 affected and 6unaffected family members. The rs143224912 variant in SETDB1 wasexcluded although it segregated with the disease phenotype, as it waspresent in 3% of the normal control population samples and due to acomplete lack of conservation of the amino acid residue altered by thechange throughout all orthologous species. The missense variantrs138355706, (229C>T, missense causing a p.R77C mutation in S100A3)segregated with ILD. All 3 affected patients were homozygous forrs138355706. Both parents were heterozygous for this variant andunaffected siblings and family members and an additional 7 unaffectedextended family members were either heterozygous for rs138355706 or werehomozygous wild-type (FIG. 1A). In addition, 500 ethnically-matchednormal controls were genotyped for this change by re-sequencing ofS100A3. Three of these individuals were heterozygous, but none werehomozygous for rs138355706, indicating a minor allele frequency of 0.3%.Sequencing of the S100A3 intronic and 5′ flanking sequences wasperformed in the affected patients and no other variants were identified(data not shown).

Haplotype Analysis.

Haplotype analysis was carried out using 8 markers (4 microsatellitemarkers flanking S100A3 and 3 further intragenic markers) confirmed thatthe three affected individuals share a specific disease haplotype onboth chromosomes that is not present in the unaffected individuals (FIG.1C). Similar analysis demonstrated all 3 normal controls that wereheterozygous for this change carry the disease haplotype on onechromosome and hence are related to the family therefore excluding themfrom the normal control set, providing additional support for the S100A3p.R77C mutation in the pathogenesis of ILD in this family.

Consequences of S100A3 c.

229 C>T mutation. The c.229 C>T mutation results in an arginine tocysteine missense at residue 77 within the second of two EF-handcalcium-binding motifs in the 103 amino acid protein. The predictedconsequences of this mutation on protein structure/function wasevaluated using the Polyphen-2 (ver. 2.2.2) and SIFT predictionprograms. Both programs predicted minor effects of the mutation onprotein structure/function with Polyphen-2 and SIFT (version 1.03)scores of 0.004 and 0.21, respectively. Since microRNAs can modifytranslation efficiency by binding to coding sequence as well as 3′UTRsequence, whether the c.229 C>T mutation altered any known microRNAbinding site using MicroSNiPer (release 19) and a minimum 7-nucleotideseed sequence and found no effect of the SNV on predicted microRNAbinding sites was analyzed.

Analysis by qRT-PCR demonstrated decreased expression of S100A3 inpatients' samples compared to control samples (FIG. 2C).

Using qRT-PCR to detect the expression of mutated and control S100A3, itwas found that the reduced protein expression was paralleled by reducedmRNA levels for mutant (HEK-M) compared to control S100A3 (HEK-W), seeFIG. 2D.

Furthermore, the effect of the mutation on the mutant protein expressionwas confirmed by experiments in which both mutant and wild type S100A3were HA-tagged and overexpressed in human normal lung fibroblasts. Usinganti-HA antibodies to detect S100A3 expression it was found that aconsistently reduced mutant S100A3 protein expression at allconcentrations of plasmid DNA (FIG. 2E), but similar mRNA levels forwild-type and mutant HA-tagged S100A3 (FIG. 2F).

Effect of S100A3 Mutation on Intracellular Calcium Homeostasis andMitochondrial Functions.

Since the S100 family of protein is calcium binding, the possibilityexisted that the mutated protein may affect calcium homeostasis.

It was found that receptor mediated calcium release was significantlyreduced in patients compared to control fibroblast (FIG. 3A).

Furthermore a clear disparity between the levels of ionomycin-inducedcalcium release in control and patients cells was demonstrated (FIG.3B).

The effect on intracellular calcium was confirmed in experiments inwhich patients fibroblasts were transfected with wild type S100A3transcripts. In this case wild-type S100A3 transcripts significantlyrestored the ionomycin-induced intracellular calcium levels to that ofcontrol levels (FIG. 3C, last bar). Furthermore normal cells transfectedwith mutant S100A3 exhibited similar calcium homeostasis to that seen inpatients' cells (FIG. 3C, third bar).

Since the mitochondria are one of the major intracellular calcium storesin many cell types, Mito Tracker® Red CMXRos was used to map possibledifferences in mitochondrial staining between control and patient'scells. Whereas control cells exhibited the “normal” distinct tubularshapes of mitochondria (FIG. 3D), patients cells exhibited more punctatefluorescence associated with aberrant mitochondrial morphology (FIG.3E).

In addition patient's cells appear to have more mitochondrial stainingthan control cells (FIG. 3F).

The integrity of the mitochondria in both patient's and control cellswas further investigated by measuring the effect of oxidative stressinduced by treating the cells with hydrogen peroxide. FIG. 3Gillustrates the ability of control cells to resist oxidative loadcompared to the patient's cells.

As shown herein, the inventors have identified a novel association forthe c. 229C>T (p.R77C) SNV in S100A3 with ILD occurring in threesiblings born to consanguineous parents of Saudi Arabian origin.Although p.R77C is classified as a SNP it has not been reported in theheterozygous or homozygous state in the European American population andis only found extremely rarely in the African American population andonly in a heterozygous form (Minor allele frequency 0.003%). Furtherscreening and exclusion of this variant in 997 ethnically matched,unrelated healthy control Saudis further confirms its rarity. Furthersequencing of the full intronic sequence and 3′ untranslated region ofS100A3 excluded any other disease-causing variations in the S100A3 genesequence in the affected family members. The mutation did not alter anypredicted microRNA binding sites that could affect protein expression.

S100A3 is a member of the S100 calcium binding proteins and contains twoEF-hand calcium-binding domains required for its function; Kizawa K,Uchiwa H, Murakami U. Highly-expressed S100A3, a calcium-bindingprotein, in human hair cuticle. Biochimica et Biophysica Acta 1996;1312:94-8. The arginine residue altered in the patients is completelyconserved among orthologs in five mammalian species and it is locatedwithin one of the two EF-calcium binding motifs of the gene, potentiallyindicating more severe consequences for the protein function than waspredicted by the Polyphen-2 and SIFT prediction programs. The missensemutation identified in S100A3 gene in our ILD patients involves asubstitution of a positively charged arginine to an uncharged cysteine(p.R77C). Previously, it has been shown that substitution of arginine byalanine at position 77 of S100A3 resulted in a 2.7-fold increase in Ca²⁺affinity in the mutant protein compared to the wild type; Kizawa K,Takahara H, Troxler H, Kleinert P, Mochida U, Heizmann C W. Specificcitrullination causes assembly of a globular S100A3 homotetramer: aputative Ca2+ modulator matures human hair cuticle. The Journal ofBiological Chemistry 2008; 283:5004-13.

These clinical, molecular and functional results show that S100A3protein is associated with pathogenesis of lung fibrosis and a goodtarget to which treatments of lung injury may be directed.

Terminology

Terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention.

The headings (such as “Background” and “Summary”) and sub-headings usedherein are intended only for general organization of topics within thepresent invention, and are not intended to limit the disclosure of thepresent invention or any aspect thereof. In particular, subject matterdisclosed in the “Background” may include novel technology and may notconstitute a recitation of prior art. Subject matter disclosed in the“Summary” is not an exhaustive or complete disclosure of the entirescope of the technology or any embodiments thereof. Classification ordiscussion of a material within a section of this specification ashaving a particular utility is made for convenience, and no inferenceshould be drawn that the material must necessarily or solely function inaccordance with its classification herein when it is used in any givencomposition.

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise.

It will be further understood that the terms “comprises” and/or“comprising,” when used in this specification, specify the presence ofstated features, steps, operations, elements, and/or components, but donot preclude the presence or addition of one or more other features,steps, operations, elements, components, and/or groups thereof.

As used herein, the term “and/or” includes any and all combinations ofone or more of the associated listed items and may be abbreviated as“/”.

Links are disabled by deletion of http: or by insertion of a space orunderlined space before www. In some instances, the text available viathe link on the “last accessed” date may be incorporated by reference.

As used herein in the specification and claims, including as used in theexamples and unless otherwise expressly specified, all numbers may beread as if prefaced by the word “substantially”, “about” or“approximately,” even if the term does not expressly appear. The phrase“about” or “approximately” may be used when describing magnitude and/orposition to indicate that the value and/or position described is withina reasonable range of values and/or positions. For example, a numericvalue may have a value that is +/−0.1% of the stated value (or range ofvalues), +/−1% of the stated value (or range of values), +/−2% of thestated value (or range of values), +/−5% of the stated value (or rangeof values), +/−10% of the stated value (or range of values), +/−15% ofthe stated value (or range of values), +/−20% of the stated value (orrange of values), etc. Any numerical range recited herein is intended toinclude all sub-ranges subsumed therein.

Disclosure of values and ranges of values for specific parameters (suchas temperatures, molecular weights, weight percentages, etc.) are notexclusive of other values and ranges of values useful herein. It isenvisioned that two or more specific exemplified values for a givenparameter may define endpoints for a range of values that may be claimedfor the parameter. For example, if Parameter X is exemplified herein tohave value A and also exemplified to have value Z, it is envisioned thatparameter X may have a range of values from about A to about Z.Similarly, it is envisioned that disclosure of two or more ranges ofvalues for a parameter (whether such ranges are nested, overlapping ordistinct) subsume all possible combination of ranges for the value thatmight be claimed using endpoints of the disclosed ranges. For example,if parameter X is exemplified herein to have values in the range of 1-10it also describes subranges for Parameter X including 1-9, 1-8, 1-7,2-9, 2-8, 2-7, 3-9, 3-8, 3-7, 2-8, 3-7, 4-6, or 7-10, 8-10 or 9-10 asmere examples. A range encompasses its endpoints as well as valuesinside of an endpoint, for example, the range 0-5 includes 0, >0, 1, 2,3, 4, <5 and 5.

As used herein, the words “preferred” and “preferably” refer toembodiments of the technology that afford certain benefits, undercertain circumstances. However, other embodiments may also be preferred,under the same or other circumstances. Furthermore, the recitation ofone or more preferred embodiments does not imply that other embodimentsare not useful, and is not intended to exclude other embodiments fromthe scope of the technology. As referred to herein, all compositionalpercentages are by weight of the total composition, unless otherwisespecified. As used herein, the word “include,” and its variants, isintended to be non-limiting, such that recitation of items in a list isnot to the exclusion of other like items that may also be useful in thematerials, compositions, devices, and methods of this technology.Similarly, the terms “can” and “may” and their variants are intended tobe non-limiting, such that recitation that an embodiment can or maycomprise certain elements or features does not exclude other embodimentsof the present invention that do not contain those elements or features.

The description and specific examples, while indicating embodiments ofthe technology, are intended for purposes of illustration only and arenot intended to limit the scope of the technology. Moreover, recitationof multiple embodiments having stated features is not intended toexclude other embodiments having additional features, or otherembodiments incorporating different combinations of the stated features.Specific examples are provided for illustrative purposes of how to makeand use the compositions and methods of this technology and, unlessexplicitly stated otherwise, are not intended to be a representationthat given embodiments of this technology have, or have not, been madeor tested.

All publications and patent applications mentioned in this specificationare herein incorporated by reference in their entirety to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference,especially referenced is disclosure appearing in the same sentence,paragraph, page, or section of the specification in which theincorporation by reference appears.

The citation of references herein does not constitute an admission thatthose references are prior art or have any relevance to thepatentability of the technology disclosed herein. Any discussion of thecontent of references cited is intended merely to provide a generalsummary of assertions made by the authors of the references, and doesnot constitute an admission as to the accuracy of the content of suchreferences.

1. A method for determining a risk of fibrosis in a person comprisingdetecting a mutant S100A3 protein in a biological sample from theperson, wherein the mutant S100A3 protein comprises an amino acidresidue other than arginine at position 77 of SEQ ID NO: 2, selecting asubject in need of treatment when the mutant S100A3 protein is detected,and treating the subject to reduce a risk of the fibrosis or treatingthe subject for the fibrosis by administering a drug, a chaperonin, or awild-type S100A3 protein.
 2. The method of claim 1, wherein the methodcomprises detecting the mutant S100A3 protein and wherein the mutantS100A3 protein comprises an amino acid residue that is cysteine at aposition corresponding to position 77 of SEQ ID NO:
 2. 3. The method ofclaim 1, wherein the method of detecting the mutant protein comprisesdetecting a polynucleotide encoding the mutant S100A3 protein.
 4. Themethod of claim 1, wherein the method comprises detecting the mutantS100A3 protein which comprises the amino acid sequence described by SEQID NO:
 4. 5. The method of claim 1, wherein the method comprisescontacting the mutant S100A3 protein with an antibody that binds to itand detecting a polynucleotide encoding the mutant S100A3 protein. 6.(canceled)
 7. (canceled)
 8. (canceled)
 9. (canceled)
 10. The method ofclaim 1 that further comprises detecting a polynucleotide that encodesthe mutant S100A3 protein using probes and/or primers that recognize thepolynucleotide comprising rs138355706 (229C>T).
 11. The method of claim1, further comprising detecting whether the subject is homozygous for apolynucleotide encoding a mutant S100A3 protein, heterozygous for apolynucleotide encoding a mutant S100A3 protein, or whether the subjectis homozygous for a gene encoding a not mutated S100A3 protein.
 12. Themethod of claim 1, wherein said fibrosis is organ fibrosis.
 13. Themethod of claim 1, wherein said fibrosis is pulmonary fibrosis.
 14. Amethod for reducing the risk of fibrosis or lung disease, or fortreating fibrosis or lung disease in a subject who has at least one geneencoding a non-functional mutant S100A3 protein comprising administeringto said subject a polynucleotide encoding a functional S100A3 protein,administering a functional S100A3 protein, or administering at least oneactivator or inhibitor of an S100A3 protein.
 15. The method of claim 14,wherein the subject has at least one gene comprising a missense mutationthat is rs138355706, (229C>T).
 16. The method of claim 14, wherein saidadministering comprises administering a polynucleotide encoding afunctional S100A3 protein into the respiratory system.
 17. The method ofclaim 14, wherein said administering comprises administering afunctional S100A3 protein into the subject's lungs and further comprisesadministering an antibody or antibody fragment that binds to the mutantS100A3 polypeptide of SEQ ID NO:
 4. 18. The method of claim 14, whereinsaid administering comprises administering at least one activator orinhibitor of S100A3 protein into the subject's lungs.
 19. (canceled) 20.(canceled)
 21. The method of claim 1, wherein said treating comprisesadministering a wild-type, functional S100A3 protein to the respiratorysystem of the subject who has the S100A3 mutation.
 22. The method ofclaim 1, wherein said treating comprises administering a polynucleotideencoding a wild-type, functional S100A3 protein to the respiratorysystem of the selected subject who has the S100A3 mutation.
 23. Themethod of claim 1, wherein said treating comprises administering atleast one chaperonin to the respiratory system of the subject who hasthe S100A3 mutation.
 24. The method of claim 1, wherein said treatingcomprises administering at least one anti-inflammatory drug to therespiratory system of the subject who has the S100A3 mutation.
 25. Themethod of claim 1, wherein said treating comprises administering anantibody that binds to a S100A3 protein to the respiratory system of thesubject who has the S100A3 mutation.